The citric acid cycle is the final common pathway for the aerobic oxidation of fuel molecules. Moreover, as we will see shortly (Section 17.4) and repeatedly elsewhere in our study of biochemistry, the cycle is an important source of building blocks for a host of important biomolecules. As befits its role as the metabolic hub of the cell, entry into the cycle and the rate of the cycle itself are controlled at several stages.

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As stated earlier, glucose can be formed from pyruvate (Section 16.3). However, the formation of acetyl CoA from pyruvate is an irreversible step in animals and thus they are unable to convert acetyl CoA back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to one of two principal fates: oxidation to CO₂ by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid (Figure 17.16). As expected of an enzyme at a critical branch point in metabolism, the activity of the pyruvate dehydrogenase complex is stringently controlled. High concentrations of reaction products inhibit the reaction: acetyl CoA inhibits the transacetylase component (E₂) by binding directly, whereas NADH inhibits the dihydrolipoyl dehydrogenase (E₃). High concentrations of NADH and acetyl CoA inform the enzyme that the energy needs of the cell have been met or that fatty acids are being degraded to produce acetyl CoA and NADH. In either case, there is no need to metabolize pyruvate to acetyl CoA. This inhibition has the effect of sparing glucose, because most pyruvate is derived from glucose by glycolysis (Section 16.1).

The key means of regulation of the complex in eukaryotes is covalent modification (Figure 17.17). Phosphorylation of the pyruvate dehydrogenase component (E₁) by pyruvate dehydrogenase kinase (PDK) switches off the activity of the complex. There are four isozymes of PDK that are expressed in a tissue-specific manner. Deactivation is reversed by the pyruvate dehydrogenase phosphatase (PDP), of which there are two isozymic forms. In mammals, the kinase and the phosphatase are associated with the E₂-E₃-BP complex, again highlighting the structural and mechanistic importance of this core. Both the kinase and the phosphatase are regulated. To see how this regulation works in biological conditions, consider muscle that is becoming active after a period of rest (Figure 17.18). At rest, the muscle will not have significant energy demands. Consequently, the NADH/NAD⁺, acetyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios promote phosphorylation and inactivation of the complex by activating PDK. In other words, high concentrations of immediate (acetyl CoA and NADH) and ultimate (ATP) products inhibit the activity. Thus, pyruvate dehydrogenase is switched off when the energy charge is high.

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As exercise begins, the concentrations of ADP and pyruvate will increase as muscle contraction consumes ATP and glucose is converted into pyruvate to meet the energy demands. Both ADP and pyruvate activate the dehydrogenase by inhibiting the kinase. Moreover, the phosphatase is stimulated by Ca²⁺, the same signal that initiates muscle contraction. A rise in the cytoplasmic Ca²⁺ level (Section 35.2) elevates the mitochondrial Ca²⁺ level. The rise in mitochondrial Ca²⁺ activates the phosphatase, enhancing pyruvate dehydrogenase activity.

In some tissues, the phosphatase is regulated by hormones. In liver, epinephrine binds to the α-adrenergic receptor to initiate the phosphatidylinositol pathway (Section 14.1), causing an increase in Ca²⁺ concentration that activates the phosphatase. In tissues capable of fatty acid synthesis, such as the liver and adipose tissue, insulin, the hormone that signifies the fed state, stimulates the phosphatase, increasing the conversion of pyruvate into acetyl CoA. Acetyl CoA is the precursor for fatty acid synthesis (Section 22.4). In these tissues, the pyruvate dehydrogenase complex is activated to funnel glucose to pyruvate and then to acetyl CoA and ultimately to fatty acids.

In people with a phosphatase deficiency, pyruvate dehydrogenase is always phosphorylated and thus inactive. Consequently, glucose is processed to lactate rather than acetyl CoA. This condition results in unremitting lactic acidosis—high blood levels of lactic acid. In such an acidic environment, many tissues malfunction, most notably the central nervous system (Problems 13 and 14).

The rate of the citric acid cycle is precisely adjusted to meet an animal cell’s needs for ATP (Figure 17.19). The primary control points are the allosteric enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, the first two enzymes in the cycle to generate high-energy electrons.

The first control site is isocitrate dehydrogenase. The enzyme is allosterically stimulated by ADP, which enhances the enzyme’s affinity for substrates. The binding of isocitrate, NAD⁺, Mg²⁺, and ADP is mutually cooperative. In contrast, ATP is inhibitory. The reaction product NADH also inhibits isocitrate dehydrogenase by directly displacing NAD⁺. It is important to note that several steps in the cycle require NAD⁺ or FAD, which are abundant only when the energy charge is low.

A second control site in the citric acid cycle is α-ketoglutarate dehydrogenase, which catalyzes the rate-limiting step in the citric acid cycle. Some aspects of this enzyme’s control are like those of the pyruvate dehydrogenase complex, as might be expected from the homology of the two enzymes. α-Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced when the cell has a high level of ATP. α-Ketoglutarate dehydrogenase deficiency is observed in a number of neurological disorders, including Alzheimer disease.

The use of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase as control points integrates the citric acid cycle with other pathways and highlights the central role of the citric acid cycle in metabolism. For instance, the inhibition of isocitrate dehydrogenase leads to a buildup of citrate, because the interconversion of isocitrate and citrate is readily reversible under intracellular conditions. Citrate can be transported to the cytoplasm, where it signals phosphofructokinase to halt glycolysis (Section 16.2) and where it can serve as a source of acetyl CoA for fatty acid synthesis (Section 22.4). The α-ketoglutarate that accumulates when α-ketoglutarate dehydrogenase is inhibited can be used as a precursor for several amino acids and the purine bases (Chapter 23 and Chapter 25).

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In many bacteria, the funneling of two-carbon fragments into the cycle also is controlled. The synthesis of citrate from oxaloacetate and acetyl CoA carbon units is an important control point in these organisms. ATP is an allosteric inhibitor of citrate synthase. The effect of ATP is to increase the value of K_M for acetyl CoA. Thus, as the level of ATP increases, less of this enzyme is bound to acetyl CoA and so less citrate is formed.

Four enzymes crucial to cellular respiration are known to contribute to the development of cancer: succinate dehydrogenase, fumarase, pyruvate dehydrogenase kinase and isocitrate dehydrogenase. Mutations that alter the activity of the first three of these enzymes enhance aerobic glycolysis (Section 16.2). In aerobic glycolysis, cancer cells preferentially metabolize glucose to lactate even in the presence of oxygen. Defects in these enzymes share a common biochemical link: the transcription factor hypoxia inducible factor 1 (HIF-1).

Normally, HIF-1 up-regulates the enzymes and transporters that enhance glycolysis only when oxygen concentration falls, a condition called hypoxia. Under normal conditions, HIF-1 is hydroxylated by prolyl hydroxylase 2 and is subsequently destroyed by the proteasome, a large complex of proteolytic enzymes (Chapter 23). The degradation of HIF-1 prevents the stimulation of glycolysis. Prolyl hydroxylase 2 requires α-ketoglutarate, ascorbate, and oxygen for activity. Thus, when oxygen concentration falls, the prolyl hydroxylase 2 is inactive, HIF-1 is not hydroxylated and not degraded, and the synthesis of proteins required for glycolysis is stimulated. As a result, the rate of glycolysis is increased.

Recent research suggestions that defects in the enzymes of the citric acid cycle may significantly affect the regulation of prolyl hydroxylase 2. When either succinate dehydrogenase or fumarase is defective, succinate and fumarate accumulate in the mitochondria and spill over into the cytoplasm. Both succinate and fumarate are competitive inhibitors of prolyl hydroxylase 2. The inhibition of prolyl hydroxylase 2 results in the stabilization of HIF-1, since HIF-1 is no longer hydroxylated. Lactate, the end product of glycolysis, also appears to inhibit prolyl hydroxylase 2 by interfering with the action of ascorbate. In addition to increasing the amount of the proteins required for glycolysis, HIF-1 also stimulates the production of pyruvate dehydrogenase kinase (PDK). The kinase inhibits the pyruvate dehydrogenase complex, preventing the conversion of pyruvate into acetyl CoA. The pyruvate remains in the cytoplasm, further increasing the rate of aerobic glycolysis. Moreover, mutations in PDK that lead to enhanced activity contribute to increased aerobic glycolysis and the subsequent development of cancer. By enhancing glycolysis and increasing the concentration of lactate, the mutations in PDK result in the inhibition of hydroxylase and the stabilization of HIF-1.

Mutations in isocitrate dehydrogenase result in the generation of an oncogenic metabolite, 2-hydroxyglutarate. The mutant enzyme catalyzes the conversion of isocitrate to α-ketoglutarate, but then reduces α-ketoglutarate to form 2-hydroxyglutarate. 2-Hydroxyglutarate alters the methylation patterns in DNA (Section 32.2) and reduces dependence on growth factors for proliferation. These changes alter gene expression and promote unrestrained cell growth.

These observations linking citric acid cycle enzymes to cancer suggest that cancer is also a metabolic disease, not simply a disease of mutant growth factors and cell cycle control proteins. The realization that there is a metabolic component to cancer opens the door to new thinking about the control of cancer. Indeed, preliminary experiments suggest that if cancer cells undergoing aerobic glycolysis are forced by pharmacological manipulation to use oxidative phosphorylation, the cancer cells lose their malignant properties. It is also interesting to note that the citric acid cycle, which has been studied for decades, still has secrets to be revealed by future biochemists.

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